Chapt07 Lecture(1)

8/11/2019 Chapt07 Lecture(1)

1/40

1Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Chapter 07

From DNA to

Ptorein


2/40

Learning Outcomes

Major components and their functions of DNA

replication, transcription and translation

Differences Between Eukaryotic and Prokaryotic

Gene Expression

How do glucose and lactose control Lac operon


3/40

Genome: complete set of genetic information

Chromosome plus technically plasmids

Functional unit is gene

Encodes gene product, usually a protein

Study of nucleotide sequence is genomics

7.1. Overview


4/40

Cells must accomplish two tasks to multiply

DNA replication

Gene expression (transcription and translation)

Information flow from DNA RNA protein

Central dogma of molecular biology

7.1. Overview

Gene Expression

Transcription

Copies the information in DNA

into RNA.

Translation

Interprets the information carried

by RNA to synthesize the

encoded protein.

DNA ReplicationDuplicates the DNA molecule

so its encoded information

can be passed on to the next

generation.

Protein

RNA

DNA

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.


5/40

7.1. Overview

PP

S

G

G

G

T

TO

PO

P O

P O

PO

O

P

O

P

O

P

O

P

DNA

3 hydroxyl3 end 5 end 5 phosphate

Nucleotide

3 hydroxyl3 end5 end5 phosphate

Base

pairs

Sugar

Sugar

Sugar

Sugar

Sugar

HO

Hydrogenbonds

S

S

S

S

O


complementaryantiparallel


6/40

RNA (ribonucleic acid)

Ribose instead of deoxyribose

Uracil in place of thymine

Usually shorter single strand

Synthesized from DNA template strand RNA molecule is transcript

Base-pairing rules apply except uracil pairs with adenine

Transcript quickly separates from DNA

Characteristics of RNA


7/40

RNA (ribonucleic acid)

Three types required for gene expression

Messenger RNA (mRNA)

Ribosomal RNA (rRNA)

Transfer RNA (tRNA)

Characteristics of RNA

DNA

Protein-encoding gene tRNA generRNA gene

Protein

Translation

Messenger RNA (mRNA) Ribosomal RNA (rRNA) Transfer RNA (tRNA)

Transcription



8/40

7.2. DNA ReplicationCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

(a)

Origin of

replication

Replication of chromosomal

DNA starts at the origin ofreplication and then proceeds

in both directions. DNA replication is semiconservative,meaning each of the two molecules

created contains one of the originalstrands paired with a newly

synthesized strand.

Newstrand

Originalstrand

New

strand

Originalstrand

DNA Replication

The replication forks ultimately

meet at a terminating site.

Site where

replicationends

Bidirectional replication createstwo advancing forks where DNA

synthesis is occurring.

Replicationforks

RNA primers


9/40

7.2. DNA Replication

Replication forksReplication forks(b)

Original

double-strandedmolecule

From J. Cairns, "The Chromosome of E. coli" in Cold Spring Harbor Symposia on Quant itative Biology, 77, Fig. 2, Pg. 44 1963 by Cold Spring Harbor Laboratory Press



10/40

DNA replication

Coordinated process

Many enzymes, proteins

Replisomes

Assembly line

The Process of DNA Replication


11/40

DNA polymerases synthesize in 5 to 3 direction

Hydrolysis of high-energy phosphate bond powers

DNA polymerase can only add nucleotides, not initiate

Require primers at origin of replication


C C C C

C C C

A A A A A A

AAAA

T T T T T T T

TTTT

T C

G G G

C

A

A

T

T

G

C

A

T

G G G

GG

5

3

3

5

T A

A

G

O O O

A

PPP

OO

P P

O

P

PP

Template strand

New strandDNA polymerase

Direction

of synthesis

OH OH

DNA Replication



12/40

Helicases unzip DNA strands

Reveals template sequences

Leading strand synthesized

continuously

Lagging strand synthesized

discontinuously

Production of Okazaki fragments


5

3

3

5

5

5

5

3

3

5

5

5

3

3

5

4

3

2

1

5

3

5

5

Replication forks

A helicase unzips

the two strands of DNA. Helicase

Leading

strand

RNA primer

DNA polymerase adds

nucleotides onto the 3end of the strand.

Okazaki fragment

of the lagging strand

Synthesis of the lagging strand must be reinitiated as more

template is exposed. Each time synthesis is reinitiated,a new RNA primer must be made. Discontinuous synthesis

generates Okazaki fragments.

Primase synthesizes

the RNA primer.

Synthesis of the leading strand

proceeds continuously as freshtemplate is exposed.

DNA ligase seals

the gaps between

Okazaki fragmentsby forming a covalent

bond between them.

DNA ligase

As DNA p olymerase adds nucleotides

to the 3 end of one Okazaki fragment,it encounters the 5 end of another.

A different type of DNA polymerase

then removes the RNA primernucleotides and simultaneously

replaces them with d eoxynucleotides.

Copyright The McGraw-Hill Companies, Inc. P ermission required for reproduction or display.


13/40

Human diseases related with DNA replication

Faulty DNA Replication Linked To Neurological Diseases:

Link Between CGG Repeats In DNA and NeurologicalDisorders

Pelizaeus-Merzbacher Disease (PMD) is an X-linked

recessive dysmyelinating disorder. Affected children show'head nodders' and 'eye waggers.' (CNS system). Due to

deletion, duplication and mutation in multiple genes.


14/40

Transcription

RNA polymerase synthesizes single-stranded RNA

Uses DNA template

Synthesizes in 5 to 3 direction

Can initiate without primer Binds to promoter

Found upstream of genes

Stops at terminator

Transcription ends

7.3. Gene Expression in Bacteria


Region transcribed

Terminator

RNA

DNA Promoter Transcription

35


15/40

Sigma () factor recognizes promoter

Subunit loosely attached to RNA polymerase

Various types recognize different promoters

Eukaryotic cells, archaea use transcription factors

Transcription


16/40


17/40

Process of decoding information in mRNA

mRNA is temporary copy of genetic information

Major components are mRNA, ribosomes, tRNAs, and

accessory proteins

Translation


18/40

Genetic code: three nucleotides = codon

Redundancy: code is degenerate

Translation


19/40

Nucleotide sequence defines coding region

Designates beginning, end of region to be translated

Translation

Gene Expression

GlnSer Met

5 3

Translation

mRNA

Ribosome-

binding site

Start

codon

Region translated

Stop

codon

Protein

Translation

Phe

Ser His

Cys TyrSer

Pro Leu

AlaTyr Glu Val

Gly

Transcription



20/40

Three reading frames possible

Depends on start of coding region

Correct reading frame is critical

Incorrect will yield different, likely nonfunctional protein

Translation

C U G G C A U U G C C U U A U



TyrProLeuLeu Ala

Trp His LeuCys

LeuAlaIleGly

Reading frame #3

Reading frame #2

Reading frame #1



21/40

Transfer RNAs (tRNAs) deliver

correct amino acid

Each has specific anticodon sequence

Base-pairs with correct codon

Each carries appropriate amino acid After delivering, tRNA can be recycled

Enzyme in cytoplasm attaches

appropriate amino acid

Translation

Pro

Pro

5

C C GG G C

3

G G C

Amino acid

Hydrogen bond

Anticodon

Codon

Anticodon

tRNA

(b)

(a)mRNA

tRNA



22/40

Translation in prokaryotes begins before

transcription is complete

Translation

Ribosome

Polypeptide

mRNA strand

Ribosome-

binding site

Start

codon

DNA

Translation

Transcription

Gene Expression

3

5

5

3

5



23/40

Initiation of Translation

Part of ribosome binds to mRNA sequence

Termed ribosome-binding site

First AUG after that site serves as start codon

Complete ribosome assembles at start codon

Initiating tRNA brings altered form of methionine

Occupies P-site

Translation

5 3

U A C

A A A A A A C A AU U UUUUG G G G GC C C G

f-Met

Initiation

The initiating tRNA, carrying the amino

acid f-Met, base-pairs with the start

codon and occupies the P-site.E-site

P-site

A-site

mRNA



24/40

Initiation of Translation (continued)

Ribosome has two sites to which tRNAs can bind

P-site occupied by tRNA carrying methionine

Another tRNA recognizes codon in empty A-site

Occupies A-site, brings correct amino acid

Translation

U A C

A A A A A A C A AU U UUUUG G G G GC C

CG G

C G

5 3

A tRNA that recognizes the next codon

then fills the unoccupied A-site.

f-Met Pro



25/40

Initiation of Translation (continued)

A-site and P-site now occupied by correct tRNAs

Enzyme creates peptide bond between their amino acids

Amino acid from tRNA in P-site added to amino acid

carried by tRNA in A-site

Translation

U A C


CG G

C G

5 3

The ribosome catalyzes the joining of theamino acid carried by the tRNA in the

P-site to the one carried by the tRNA in

the A-site.

f-Met

Pro

(a)

Peptide bond



26/40

Elongation of Polypeptide Chain

Ribosome advances along mRNA in 5 to 3 direction

Initiating tRNA exits through E-site

Remaining tRNA carrying both amino acids occupies P-site

A-site transiently empty

A tRNA that recognizes codon in A-site quickly attaches

Translation

ElongationThe ribosome advances a distance ofone codon. The tRNA that occupied the

P-site exits through the E-site and the

tRNA that was in the A-site occupies theP-site. A tRNA that recognizes the next

codon quickly fills the empty A-site.

Ribosome moves along mRNA.


CG G

C G

E-site

P-site

A-site

f-Met

Pro

5 3



27/40

Elongation of Polypeptide Chain (continued)

Peptide bond formed between amino acids

Ribosome advances one codon on mRNA

tRNA exits E-site, new tRNA occupies A-site

Process repeats

Once ribosome clears initiating sequences, another

ribosome can bind: polyribosome, or polysome

Translation

The ribosome continues advancingdown the mRNA in the 5 to 3 direction,

moving one codon at a time.

A A A A A A C A AU U UUUUG G G G GC CGA UC G

5

(b)

3

f-Met

Pro

Tyr



28/40

Termination

Elongation continues until ribosome reaches stop codon

Not recognized by tRNA

Enzymes free polypeptide

Break covalent bond joining to tRNA

Translation

Termination

Translation continues until a stop

codon is reached, signaling the end

of the process. No tRNA molecules

recognize a stop codon.


GA U

C G

f-Met ProTyr

Asp

Tyr

Glu

35



29/40

Termination (continued)

Freed ribosome falls off mRNA

Disassociates into component subunits (30S and 50S)

Subunits can be reused to initiate translation at other sites

Translation

The components dissemble, releasingthe newly formed polypeptide.

(c)

f-MetPro Tyr

GluAsp

Tyr


Aminoglycoside antibiotics allows incorporation of an amino acid and permits

translation to read through premature stop codons.

Treat 510% of cystic fibrosis patients premature termination codons in the

CFTR gene.

7 4 Differences Between Eukaryotic and Prokaryotic


30/40

Eukaryotic transcription, translation differs

mRNA synthesized in precursor form: pre-mRNA

Must be processed during and after transcription

5 end capped with methylated guanine derivative

Binds specific proteins: stabilize, enhance translation

3 end modified via polyadenylation

Addition of ~200 adenine derivatives to new 3 end

Poly A tail stabilizes transcript, enhances translation

7.4. Differences Between Eukaryotic and Prokaryotic

Gene Expression

7 4 Differences Between Eukaryotic and Prokaryotic


31/40

Eukaryotic transcription, translation differs (cont)

7.4. Differences Between Eukaryotic and Prokaryotic

Gene Expression

Splicing removes introns

Non-coding sequences

Exons are expressed regions

mRNA transported to

cytoplasm

mRNA typically monocistronic

Ribosomes are 80S

40S and 60S subunits

Important difference for

targeting with antibiotics

Eukaryotic DNA contains

introns, which interrupt

coding regions (exons).

Transcription generates

pre-mRNA (precursor mRNA)

that contains introns. A cap

and poly A tail are then added.

Poly A tailCap

Pre-mRNA

Splicing removes introns to

create functional mRNA.

mRNA is transported out of

the nucleus to be translated

In the cytoplasm.

mRNA

Eukaryotic DNA

Exon Intron Exon Intron Exon


7 5 Sensing and Responding to Environmental


32/40

Signal Transduction

Transmits information from outside cell to inside

Allows cells to monitor and react

Quorum Sensing

Some organisms can sense density of their population Allows cells to activate genes useful with critical mass

E.g., biofilm formation, pathogens infective process

7.5. Sensing and Responding to Environmental

Fluctuations

When few cells are present, the

concentration of the signalingmolecule is low.

Bacterial cell Signaling

molecule

When many cells are present, the

signaling molecule reaches aconcentration high enough to inducethe expression of certain genes.




33/40

Two-Component Regulatory Systems


Fluctuations

Membrane-spanning sensor

Modifies internal region in response to

specific environmental variations

Phosphorylates amino acid

Response regulator

Phosphate group transferred from

sensor

Regulator turns genes on or off in

response

Examples include E. coli using nitrate

as terminal electron acceptor;

pathogens sensing magnesium levels

to recognize if within host cell

P

P

The sensor protein spans the cytoplasmic membrane.The response regulator is a protein inside the cell.

Environmental stimulus

Sensorprotein

Responseregulator

In response to a specific change in the environment, the

sensor phosphorylates a region on its internal portion.

The phosphate group is transferred to the responseregulator, which can then turn genes on or off, dependingon the system.




34/40

Natural selection can play role in gene expression

Expression of some genes changes randomly in cells Enhances survival of at least part of population

Antigenic variation is alteration of characteristics ofsurface proteins

Allows pathogens to stay one step ahead of host defenses

Phase variation involves switching genes on and off


Fluctuations

6 B i l G R l i


35/40

Genes can be routinely expressed or regulated

Regulated genes transcribed as single polycistronic

messages termed operon

E.g., lac operon for lactose metabolism

Separate operons controlled by single regulatory

mechanism constitute regulon

Often controlled by two-component regulatory systems

Global control is simultaneous regulation of numerous

genes

7.6. Bacterial Gene Regulation

Th l O M d l


36/40

Lactose and the lac Operon

No lactose: repressor prevents transcription

Lactose present: some converted to inducer allolactose

Binds to repressor

Repressor releases

operator

RNA polymerase

transcribes operon

Only occurs when

glucose unavailable

The lac Operon as a Model

No lactose in the cell

The repressor binds to operator, blocking transcription.

DNA

RNA polymerase

bound to promoter

Repressor bound

to operator

Lactose present in the cell

Some lactose is converted to allolactose. This binds to

the repressor and alters its shape, so that it can no

longer bind to the operator. If glucose is not available,

the operon will be transcribed.

Transcription

Transcription

Translation

lacAlacYlacZ

(transacetylase)(permease)(

-galactosidase)

Allolactose Non-functional

repressor

Terminator


Th l O M d l


37/40

Glucose and the lac Operon

Carbon catabolite repression (CCR) prevents expression

of lactose in presence of glucose

Prioritize carbon/energy sources; yields diauxic growth

Glucose transport system senses glucose

Catabolite activator protein (CAP)

required for transcription

Functional only when bound

by inducer cAMP

cAMP made when glucose low Inducer exclusion: lactose

transporter blocked during

glucose transport



Numberofcells(lo

garithmicscale)

Growth on

lactose

Lactose

used up

Glucose

used up

Growth on

glucose

Glucose and

lactoseadded

Time of incubation (hr)

Th l O M d l


38/40

Glucose and

the lac Operon(continued)


P

P P P P

P

P

P

P

R

R

R

R

R

R

Positive regulation of the lacoperon

Low glucoseThe phosphorylated form of the glucose transporter component activates the

enzyme that produces cAMP, which binds to the activator (CAP). The complex

of CAP and cAMP can then bind to the activatorbinding site of thela c

operon,permitting transcription. Note that lactose must be present for transcription to

occur (see figure 7.23).

High glucoseThe unphosphorylated form of the

glucose transporter componentprevents the lactose transporter

(permease) from functioning. Because

lactose cannot be moved into the cell,the inducer (allolactose) cannot

accumulate, so transcription will be

blocked (see figure 7.23).

Lactose

transporter

(permease)

Lactose

Inducer exclusion

cAMP (inducer)

ATP

E. Coli cell

Unphosphorylated

transporter component

Glucose transporter as a sensorHigh glucose

Glucose transporter as a sensorLow glucose

Glucose

Glucosetransporter

Functionalactivator

CAP

(inactive)

The unphosphorylated form of the

glucose transporter component indicates

that glucose is available in the medium.This is because the phosphorylated form

donates its phosphate group during thetransport process.

The phosphorylated form of the

transporter component indicates thatglucose is not available in the medium.

This is because it cannot donate its

phosphate during glucose transport.

Glucose


+

7 7 E k ti G R l ti


39/40

Eukaryotic regulation more complicated

Variety of approaches

Modification of chromosome structure

Regulation of initiation of transcription

Altering pre-mRNA processing and modification RNA interference (RNAi)

7.7. Eukaryotic Gene Regulation

7 7 E k ti G R l ti


40/40

RNA interference (RNAi)

Short RNA strand joins multi-protein unit

RNA-induced silencing complex (RISC)

RNA strand serves as probe for binding to mRNA

Tags mRNA for destruction

Enzymes in RISC destroy

RISC is catalytic

Rapidly silences transcripts

microRNA (miRNA) and

short interfering RNA (siRNA)About 2 dozen nucleotides;

produced differently

7.7. Eukaryotic Gene Regulation

Binding of the RNA in the RISC to

mRNA tags the mRNA for

destruction. Enzymes cut mRNA;

RISC can then bind to another

mRNA molecule.

RNA-induced silencingcomplex (RISC)

Cell produces short single-stranded

RNA.

An RNA-induced silencing complex

(RISC) assembles.


Date post:	03-Jun-2018
Category:	Documents
Upload:	emeza112
View:	220 times
Download:	0 times

Chapt07 Lecture(1)

Documents